Antagonistic Bioactivity of an Endophytic Bacterium Isolated from Epimedium Brevicornu Maxim

Full Length Research Paper

Antagonistic bioactivity of an endophytic bacterium isolated from Epimedium brevicornu Maxim

Rong-lin He1, Guo-ping Wang2, Xiao-Hong Liu1, Chu-long Zhang1* and Fu-cheng Lin1*

1State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China. 2Dayang Chemical CO. Ltd., Chaoyang Road 22, Dayang town, Jiande, 311616, China.

Accepted 2 January, 2009

Endophytic bacteria are one of the most potential biological control agents in plant disease protection. The aim of this work was to evaluate the antimicrobial activities of a strain of endophytic bacterium which was isolated from Epimedium brevicornu Maxim. The dual tests revealed that this endophytic bacterium strain displayed a wide-spectrum antimicrobial activity against 14 fugal phytopathogens and one bacterial phytopathogen; it especially strongly inhibited Alternaria alternata, Sclerotinia sclerotiorum, Verticillium dahliae, Botrytis cinerea and Botrytis fabae. Identification of this strain based on morphology, physiological and chemical characteristics and 16S rRNA gene sequence analysis demonstrated that it belonged to the genus Phyllobacterium.

Key words: Endophytic bacteria, antimicrobial bioactivity, 16S rRNA gene.

INTRODUCTION

Endophytes, by definition, are microorgnasims (mostly mechanisms of endophytic bacteria include the produc- fungi and bacteria) which inhabit in healthy living plant tion of second metabolites, nutrient and ecological niche tissues for all or part of their life cycle without causing competition, and the induction of systemic acquired apparent harmful symptoms to the host (Sturz et al., resistance of the host (Sturz et al., 2000; Kong and Ding, 2000; Wellington and Marcela, 2004). In resent years, 2001). In fact, some species of Pseudomonas and much research has focused on the bioactivities of Bacillus species have already been successfully used as endophytic fungi and endophytic bacteria (Strobel, 2003). biological control agents (Braun-Kiewnick et al., 2000; Because of living in a relatively steady environment – Cartwright et al., 1995; Kudryashava et al., 2005 Shoda, within plant tissues, endophytes may be much more 2000). Therefore, searching for new endophytic bacteria bioactive than rhizosphere microbes or any other microb- is a way of controling plant diseases by biocontrol es which were isolated from plant surface or soil are methods. (Dowler and Waiver, 1974; Andrews, 1992). As a member In this paper, an isolate of endophytic bacterium from of Endophytes, endophytic bacteria have also received Epimedium brevicomum Maxim was screened for its more attention on their antagonistic bioactivities including antimicrobial activity in vitro. In the dual culture analysis, biological control of plant diseases, plant growth stimula- it showed a broad-spectrum antimicrobial activity against tion, nitrogen-fixing and so on (Harris et al., 1994; Chen 14 stains of fungal as well as one stain of bacterial phy- et al., 1995; Graner et al., 2003; Cui et al., 2003; Qiao et topathogen; it especially strongly inhibited the growth of al., 2006). Alternaria alternata and Sclerotinia sclerotiorum. Finally, Nowadays, it is known to all that excess use of the endophytic bacterium strain EBBLQ01 was identified fungicides leads to severe large-scale pollution. With in- through morphology, physiological and chemical charac- creasing environmental awareness, developing biological teristics and homology of 16S rRNA gene sequence by strategies using endophytic bacteria are new alternatives NCBI program BLAST and phylogenetic tree analysis. to solving the contamination problems. The biocontrol

MATERIAL AND METHODS

Test phytopathogens and media *Corresponding author. E-mail: [email protected], [email protected], Tel: +86 571 8697 1185. 16 strains of fungal and 3 strains of bacterial phytopathogens for an- 192 Afr. J. Biotechnol.

timicrobial activities assays were used in this study. The tested ther identification, 16S rDNA sequence analysis was conducted. isolates; Alternaria alternata, Aspergillosis, Botrytis cinerea, Genomic DNA of EBBLQ01 was extracted as follows: a single Rhizoctonia solani, Sclerotium rolfsii, Pythium ultimum, Fusarium colony of the bacterial strain grown on NA for 2 days was picked out oxysporum f.sp cucumber, Colletotrichum orbiculare, Verticillium with a sterilized toothpick and suspended in an eppendorf tube with dahliae, S. sclerotiorum, Pestalotia diospyri, Paecilomyces 50 µl TE buffer. The tube was heated in boiled water for 5 min, and Lilaciaus, Pyricularia oryzae, Botrytis fabae, Didymella bryoniae, centrifuged at 12000 rpm for 5 min. The supernatant was removed Chryseobacterium sp., Pseudomonas solanacearum and Pseudo- to a new eppendorf tube and used as DNA template. The 16S rRNA monas cachrymans were all stored in our laboratory. Potato gene sequence was amplified by primers BSF8/20 (5'- dectrose agar (PDA) was used for antagonistic tests and fungal AGAGTTTGATCCTGGCTCAG-3'), BSR1541/20 (5'- phytopathogens maintaining. Nutrient agar (NA) was used for AAGGAGGTGATCCAGCCGCA-3'). The amplification conditions bacteria culture. was performed as the following program: initial denaturation (5 min at 94°C), 35 cycles of denaturation (60 s at 94°C), annealing (30 s at 55°C) and extension (90 s at 72°C), and final extension (10 min Isolation of the endophytic bacterium strain at 72°C). PCR product was checked by gel electrophoresis in 1.0% The healthy plant materials of E. brevicomum Maxim were collected agarose gel stained with ethidium bromide (EB). The resulting PCR from Longquan, Zhejiang province, China in May 2008. Then the product was purified by DNA gel extraction kit (Axygen, USA) and plant samples were placed in a plastic bag within an ice box and the purified PCR product was used directly for sequencing. transported to the laboratory as soon as possible. Alignments between the sequences were performed by using Samples were flushed with running water to remove the soil. The CLUSTAL W software (Thompson et al., 1997) and a phylogenetic stems and roots were cut into 10 × 10 mm pieces and treated with tree was made using PAUP version 3.1.1 (Swofford, 1993). 75% ethanol for 1 min, 5.2% NaClO for 3 min, and then three rinses in sterilized distilled water. The inner part of the root tissues were all placed on water agar (WA) medium (containing 60 µg/ml RESULTS streptomycin and 100 µg/ml ampicillin) and the plates were incubated at 25°C for 3 - 7 days. After incubation, the endophytic bacterial colony appeared and showed antimicrobial activity against Antimicrobial tests and scanning electron other isolates of endophytic fungi which had been isolated at the microscopy observation same time. The bacterial colony was picked out, streaked on NA and incubated at 28°C for 2 days to get the pure culture. After The bacterial strain EBBLQ01 was isolated from E. purification, the bacterial isolate (named as EBBLQ01) was cultivated in 5 ml of NA liquid medium with constant shaking at brevicornu Maxim. It was resistant to streptomycin and 28°C for 2 days. The culture was suspended in 20% glycerol ampicillin and depressed the growth of the endophytic solution and stored at -80°C. fungi. In the dual tests, EBBLQ01 produced inhibition zones against 14 strains of phytopathogenetic fungi and one strain of phytopathogenetic bacterium. The inhibition Antimicrobial activity assay and scanning electron microscopy zones against A. alternata, S. sclerotiorum, V. dahliae, B.

The inoculum of EBBLQ01 was prepared from cultures incubated in fabae, B. cinerea and Rhizopus sp. were larger than 20 NA liquid medium at 28°C for 3 - 5 days with constant shaking. 20 mm. We also found that the bioactivities of ferment ml of the fresh liquid culture was centrifuged at 3000 rpm for 15 min aliquots were weaker than that of the liquid endophytic at 4°C. The supernatant was removed to a new centrifuge tube and bacterial cultures (Table 1). filter sterilized. Both the bacterial inoculum and supernatant were After analysis of antagonistic activity, S. sclerotiorum used for the dual tests. which was intensively inhibited by EBBLQ01 was picked For the antifungal assay, a plug of mycelium of each fungus (5 mm diameter) was plated at the centre of the Petri dish containing 25 ml out as the model of studying the mechanism of its PDA and 50 µl liquid endophytic bacterial cultures or supernatant antimicrobial activity. In the dual test plate, the inhibition aliquots were put into the holes (5 mm diameter) 3 cm away from zones were obvious and the diameters were 23 mm the centre. All the tested plates were incubated at 25°C for 5 - 7 (Figure 1A). In the SEM photographs of confront culture days and the inhibition effects were evaluated by measuring the (Figure 2A), it could be seen that the hyphae of S. diameters of the inhibition zones. For the antibacterial assay, the bacterial pathogens were sclerotiorum first swelled then ruptured, and the cyto- cultivated in Erlenmeyer flask with 100 ml liquid NA medium at 28°C plasm started to extravagate outside. In contrast, the for 2 - 3 days. After being diluted 1000 times, the bacterial cultures hyphae were straight and thrived in normal growth were spread on the Petri dishes containing 25 ml NA. The following conditions (Figure 2B). procedures were carried out as described previously. All the tested plates were incubated at 28°C for 24 h days and then the diameters of the inhibition zones were observed. All the experiments above were repeated at least 3 times with 3 replications and sterilized Identification of the endophytic bacterium EBBLQ01 distilled water was used as CK. For scan electron microscopy (SEM), the samples observations were performed as described The colony of EBBLQ01 on LB was yellowish, smooth everywhere (Liu et al., 2007). and opaque. The thallus was rod-shaped and motiled by means of a polar flagellum (Figure 1B). EBBLQ01 was characterrized as Gram-negative, catalase-positive, me- Characterization of the endophytic bacterium strain EBBLQ01 thyl red reaction (MR) positive, Voges-Proskauer (V-P) The physiological and biochemical tests were carried out as reaction positive, oxidative metabolism of glucose posi- described for preliminary characterization (Knösel, 1984). For fur- tive and was capable of producing H2S. It hydrolyzed starch He et al. 193

Table 1. Antimicrobial activities of EBBLQ01 against phytopathogens.

Inhibition zone diameters (mm) Phytopathogen Fermentation aliquot Bacterial culture Alternaria alternata 16 26 Botrytis cinerea 12 22 Botrytis fabae 13 23 Colletotrichum orbiculare 11 19 Didymella bryoniae 9 16 Fusarium oxysporum f.sp cucumerium 9 12 Pestalotia diospyri 11 16 Pythium ultimum / / Rhizoctonia solani 11 17 Paecilomyces Lilaciaus 8 13 Rhizopus sp. 13 22 Sclerotinia sclerotiorum 14 23 Sclerotium rolfsii / / Verticillium dahliae 14 23 Penicillum digitatum 13 19 Aspergillosis sp. 11 16 Chryseobacterium sp. 12 17 Pseudomonas lachrymans - - Pseudomonas solanacearum - -

Figure 1. (A) Inhibition effect of EBBLQ01 against Sclerotinia sclerotiorum. (B) Thallus of EBBLQ01. They are arrayed in pairs, 1.1 m × 2.2 m in size.

and Tween 20, and could grow on basal medium, yeast- genus Phyllobacterium. The resulting phylogenetic tree mannitol agar, peptone agar, glucose-yeast extract agar (Figure 3) showed that EBBLQ01 and P. myrsinacearum and nutrient agar. Growth in the presence of NaCl clustered together within one subclade with a bootstrap concentrations was up to 5% and the optimal tempera- support of 96%. Taken together this suggested that strain ture of its growing was 30 - 37°C. EBBLQ01 belonged to different species. For further identification of EBBLQ01, we amplified the 16S rRNA gene sequence and compared the sequence with sequences from GenBank using BLAST program DISCUSSION (Altschul et al., 1990). The 16S rRNA gene sequence of the bacterium strain showed 99% identity to that of Previous studies demonstrated that some species of Phyllobacterium myrsinacearum. It was indicated that this Phyllobacterium sp. are resistant to many kinds of strain was phylogenetically related to members of the antibiotics (Mahdhi et al., 2007). In the present study, one 194 Afr. J. Biotechnol.

Figure 2. Hyphae of Sclerotinia sclerotiorum (A) hyphae in dual test plate was wrinkle and the cytoplasm was concentrated. (B) Normal hyphae.

Figure 3. Phylogenetic relationships of the 16S rRNA gene from EBBLQ01 (GenBank No. FJ178785) and representative related strains from GenBank. Escherichia coli (FM207521) was used as outgroup. Percentages above the branches were the frequencies which had been given in 1000 bootstrap replications.

isolate of endophytic bacterium EBBLQ01 was resistant 16S rRNA gene sequence analysis, strain EBBLQ01 was to streptomycin and ampicillin which was consistent with designed as Phyllobacterium sp. Because we only the former research. performed preliminary physiological and biochemical On the basis of physiological and chemical tests and characteristics tests and sequenced only 16S rRNA gene, He et al. 195

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